Applicant: Prof. Arndt Rolfs [arndt.rolfs@med.uni-rostock.de]
Leiter des Neurobiologischen Labors der Klinik für Neurologie und Poliklinik der Universität Rostock, Gehlsheimer Str. 20, PF 100888, 18055 Rostock,

We have demonstrated the possibility to couple different functional proteins on special sequences from the heavy chain fragment (TTC) from the tetanus toxin protein that allow a highly specific and efficient internalization of such a hybrid protein in neurons. Such a construct will mainly be of great usage in therapeutic approaches of the myelon, like in SPG-

The main features of tetanus toxin are ascribed to the effect of the toxin on the CNS. To exercise its pathogenetic action, the toxin must be transported from its site of introduction to the site of action, namely, the spinal cord. The toxin is taken up by nerve terminals of all peripheral neurons (motor, sensory, adrenergic). The accumulation of the toxin in presynaptic nerve endings that impinge on motoneurons results in blockade of inhibitory neurotransmitter release with a concomitant central disinhibition. It is clear that binding, internalization and retrograde transport of the tetanus toxin molecule are prerequisites for expression of biological activity. Furthermore, tetanus toxin has been found to be transported in all kinds of neurons (Stoeckel et al., 1975).

The tetanus toxin molecule can be cleaved by a variety of techniques to give two principal polypeptides: a fragment (ca. 100kd) composed to the light chain attached to the amino terminus of the heavy chain, and a fragment (ca. 50kD) composed to the carboxy terminus of the heavy chain. The latter has been given various names including fragment C (TTC), fragment IIc, and fragment B-IIb. There is no evidence that these fragments have important functional differences. Retrograde transport of TTC was found to occur in adrenergic, sensory and motoneurons (Bizzini et al., 1977). This latter result led the investigators to speculate that such fragments could be used to carry chemotherapeutic agents or drugs of research interest to the CNS (Bizzini et al., 1977). Subsequently, it was demonstrated that TTC could be covalently complexed to the remainder of the toxin molecule and TTC would convey the complex retrogradely from the medial rectus muscle to the oculomotor nucleus. That portion of the toxin molecule that did not contain TTC did not undergo retrograde transport (Bizzini et al., 1980). Morris et al. (1980) reported that TTC bound quite well to neural membranes and was transported retroaxonally as effectively as intact toxin.

Firstly in 1995 Francis and co-workers described the usage of a TTC-hybrid protein for the targeted delivery of a given protein demonstrated with the example of CuZn superoxide dismutase (SOD-1). Increased levels of SOD-1 are cytoprotective in experimental models of neurological disorders associated with free radical toxicity (e.g. stroke). The authors produced a recombinant, hybrid protein in E. Coli tandemly joining human SOD-1 to TTC. This approach has been published by the same group (Francis et al., 1997) demonstrating the cerebroprotective properties of human SOD-1 incomparison to SOD:Tet451. Following 2h of temporary middle cerebral artery occlusion, rats infused with equivalent activities of either SOD-1 or SOD:Tet451 for the initial 3h of reperfusion showed reductions in cerebral infarct volume of 43% and 57%, resp., compared to saline-treated. The group speculated that the cerebroprotective effects of SOD-1 may be anhanced by nueronal targeting as seen with the hybrid protein SOD:Tet451.

The group of Coen and co-workers (1997) investigated the potential use of the TTC-fragment as an in vivo neurotropic carrier. They demonstrated that a hybrid protein encoded by the lacZ-TTC gene fusion retains the biological functions of both proteins in vivo – i.e. retrograde transsynaptic transport of the TTC fragment and ß-galactosidase activity. After intramuscular injection, enzymatic activity could be detected in motoneurons and connected neurons of the brainstem areas. To the authors opinion, this strategy may be used to deliver a biological activity to neurons from the periphery to the central nervous system.